1. Field of the Invention
The present invention provides a method for treating wounds and diseases in mammals, caused by mammalian cells involved in an inflammatory response, by altering indigenous in vivo levels of peroxynitrous acid, and salts thereof. The method comprises contacting the mammalian cells with a therapeutically effective amount of a reactive oxygen species mediator, wherein the reactive oxygen species mediator is selected from the group consisting of pyruvates, pyruvate precursors, α-keto acids having four or more carbon atoms, precursors of α-keto acids having four or more carbon atoms, and the salts thereof, wherein mediation of hydrogen peroxide results in mediation of peroxynitrous acid. The present invention further provides a pharmaceutical composition for treating wounds and diseases in mammals, caused by mammalian cells involved in an inflammatory response, by altering indigenous in vivo levels of peroxynitrous acid, and salts thereof.
2. Description of the Prior Art
The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference and, for convenience, are referenced in the following text and respectively grouped in the appended bibliography.
A number of diseases are typically characterized by a marked inflammation at the site of the injury. This inflammatory process leads to further destruction of surrounding healthy tissue, and a continuation and expansion of the sites of inflammation. The over production of oxygen radicals such as hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) have been shown to activate both IkappaB kinase β (IKK-β) and Nuclear Factor kappa B (NF-kappa-B), both of which activate the inflammatory process in numerous diseases including cancer. Oxygen radicals, and the over-expression of inflammatory mediators controlled by NF-kappa-B will delay healing and destroy many of the medications used to treat the disease state.
Recently, it has been shown that deactivating a protein called IKK-β inside the cell stops cancer progression along with inflammation. IKK-β normally plays a role in healing (37,38,64) and directly activates NF-kappa-B. During an injury or infection, immune system molecules such as oxygen radicals, which deplete glutathione, can over-activate IKK-β (37,64). Once stimulated, IKK-β maintains cells alive and growing and can also promote inflammation in damaged tissues. IKK-β is also known to be elevated in infected epithelial cells in wounds and in diseases, including cancer. IKK-β also assists tumor growth in different types of cells by subverting the programmed cell death that would otherwise prevent tumor formation. Thus, IKK-β promotes tumor development and growth through inflammation.
IKK-β works by activating NF-kappa-B (39,40,64). NF-kappa-B resides in the cytoplasm as an inactive dimer, consisting of two subunits, bound to an inhibitory protein (44,49,50). The inhibitory protein is degraded in response to various environmental stimuli, such as pro-inflammatory cytokines, viruses, and oxygen radicals. This degradation allows NF-kappa-B to translocate to the nucleus where it activates genes that play a role in the regulation of inflammatory responses, including genes that encode pro-inflammatory cytokines such as the interleukins (IL) including IL-2, IL-6, IL-11, and IL-17, and tumor necrosis factor (TNF).
TNF-α functions by inducing telomerase activity in the cytoplasm of cells. NF-kappa-B also regulates nitric oxide synthetase, and genes that inhibit apoptosis which play a major role in tumor growth and survival (44,49,50). NF-kappa-B also activates telomerase transcription. Telomerase repairs shortened telomere ends on chromosomes, which makes cells immortal (such as cancer cells). NF-kappa-B, when activated, stops programmed cellular death, activates other inflammatory mediators, and increases nitric oxide synthesis and production (38-40,44,64). In patients with skin diseases including infected sites and cancer, NF-kappa-B activation is exaggerated as are other inflammatory components (37,38,44). Hydrogen peroxide and other oxygen radicals, such as peroxynitrite, activate NF-kappa-B as does cellular glutathione depletion (42-45,64).
Oxygen radicals also damage p53, a protein that inhibits tumor growth. The function of p53 is to ensure that every time a cell divides, each of the two daughter cells gets an undamaged copy of the original set of genes, free of mutations. If a cell contains damaged DNA, the p53 protein stops cellular division. Only when repairs are complete, will p53 permit DNA replication to begin. If the damage is too extensive to repair, p53 blocks the cell from dividing and commands the cell to die (34). The p53 protein triggers the process of programmed cell death. In 50-80% of all cancers, p53 is damaged and does not function (34). Thus any molecule that can regulate the over-expression of peroxynitrite while protecting nitric oxide would deactivate either IKK-β or NF-kappa-B. This regulation would inhibit inflammation that could lead to tumor development and survival (41) and enhancement of the healing process of infected and noninfected wounds, with and without drugs. Antioxidants have been shown to neutralize oxygen radicals, thus inhibit NF-kappa-B activation to inhibit inflammation and to protect DNA and proteins like p53 from oxidative damage (75) thus facilitate the healing process. Antioxidants such as vitamin C, vitamin A, acetylcysteine, vitamin E, glutathione, and pyruvate down regulate and inhibit NF-kappa-B by the reduction of oxygen radicals (40-52,55,64). High levels of nitric oxide also inhibit NF-kappa-B. Thus a technology that can regulate the production of peroxynitrite and reduce NF-kappa-B induced inflammation and protect drugs needed to treat various diseases would be very useful in the therapeutic area.
Wounds are internal or external bodily injuries or lesions caused by mechanical, chemical, viral, bacterial or thermal means, which disrupt the normal continuity of structures. Such bodily injuries include contusions, which are wounds in which the skin is unbroken; incisions, i.e., which are wounds in which the skin is broken by a cutting instrument; and lacerations, which are wounds in which the skin is broken by a dull or blunt instrument.
Wound healing consists of a series of processes whereby injured tissues are repaired, specialized tissue is generated, and new tissue is reorganized. Wound healing consists of three major phases: (a) an inflammation phase (0-3 days); (b) a cellular proliferation phase (3-12 days); and (c) a remodeling phase (3-6 months).
During the inflammation phase, platelet aggregation and clotting form a matrix which traps plasma proteins and blood cells to induce the influx of various types of cells. It is at this time that peroxynitrite is produced and causes NF-kappa-B to be over-expressed, thus delaying the healing process. This over-expression of peroxynitrite can also destroy drugs needed to treat various diseases including infected wounds. During the cellular proliferation phase, new connective or granulation tissue and blood vessels are formed. During the remodeling phase, granulation tissue is replaced by a network of collagen and elastin fibers leading to the formation of scar tissue. Most wounds also produce pain, swelling, itching, ischemia, crusting, erythema, and scarring, which is caused by the over-expression of NF-kappa-B, as a result of the over-expression of peroxynitrite. Many of these adverse side-effects are caused by the reaction of over-expressed peroxynitrite with therapeutic drugs to produce undesirable metabolites.
When cells are injured or killed as a result of a wound, a wound-healing step is desirable to resuscitate the injured cells and produce new cells to replace the dead cells. Wounds require low levels of oxygen in the initial stages of healing to suppress oxidative damage and higher levels of oxygen in the later stages of healing to promote collagen formation by fibroblasts.
Wounds produce oxygen radicals. Mammalian cells are continuously exposed to activated oxygen species such as superoxide (O2−), hydrogen peroxide (H2O2), hydroxyl radicals (OH), peroxynitrite (ONOO−) and singlet oxygen (1O2). In vivo, these reactive oxygen intermediates are generated by cells in response to aerobic metabolism, catabolism of drugs and other xenobiotics, ultraviolet and x-ray radiation, and the respiratory burst of phagocytic cells (such as white blood cells) to kill invading bacteria and viruses introduced through wounds. The toxic by-products generated form the catabolism of drugs to treat infected and cancerous cells activate the inflammatory process generally through the activation of NF-kappa-B, which can delay healing. Hydrogen peroxide, for example, is produced during respiration of most living organisms especially by stressed and injured cells.
These active oxygen species can injure cells. An example of such damage is lipid peroxidation which involves the oxidative degradation of unsaturated lipids. Lipid peroxidation is highly detrimental to membrane structure and function and can cause numerous cytopathological effects including the activation of NF-kappa-B. Cells defend against lipid peroxidation by producing radical scavengers such as superoxide dismutase, catalase, and peroxidase. Injured cells have a decreased ability to produce radical scavengers. Excess hydrogen peroxide, especially peroxynitrite, can react with DNA to cause backbone breakage of the DNA, produce mutations, and alteration and liberation of the bases. Hydrogen peroxide can also react with pyrimidines to open the 5, 6-double bond, which inhibits the ability of pyrimidines to hydrogen bond to complementary bases. Such oxidative biochemical injury can result in the loss of cellular membrane integrity, reduced enzyme activity, changes in transport kinetics, changes in membrane lipid content, leakage of potassium ions, amino acids, other cellular material, and the formation of excess keloid and scar formation.
Hydrogen peroxide also markedly potentiates the cytotoxic effects of eosinophil derived enzymes such as 5,8,11,14,17-eicosapentaenoic acid (1-4,64-67). Excess superoxide anions and hydrogen peroxide, and their by-products, specifically peroxynitrite, produced during the inflammatory phase of an injury, will destroy healthy tissue surrounding the site (19). Peroxynitrite injures membranes allowing infections to spread. Oxygen radicals can also initiate lipid peroxidation employing arachidonic acid as a substrate producing prostaglandins and leukotrienes. Hydrogen peroxide can induce arachidonic acid metabolism in alveolar macrophages (10,11,19). Hydrogen peroxide, and other oxygen radicals such as nitrogen dioxide and peroxynitrite, also activate NF-kappa-B as does cellular glutathione depletion (42-44,55,64,67-72). Oxygen radicals lower cellular levels of glutathione. Oxygen radicals also damage p53, a protein central to the inhibition of tumor growth and needed by cells to facilitate healing and DNA repair. Oxygen radicals also produce 8-isoprostanes, which are potent renal and pulmonary artery vasoconstrictors, bronchoconstrictors, and airflow obstructers (19,20,64,66-69). Oxygen radicals, and other inflammatory mediators, specifically expressed from NF-kappa-B, will reduce the efficacy and duration of drugs needed to treat infected and noninfected wounds and cancer drugs.
Excess nitric oxide, which becomes peroxynitrite, has been implicated in inflammation (26,28,29,70). This is because nitric oxide can become a toxic oxidant when it reacts with excess oxygen radicals such as hydrogen peroxide to produce nitrogen dioxide (NO2) (1-3) and peroxynitrite (ONOO). Oxygen radicals are produced by many cell types including epithelial cells, macrophages, leukocytes, monocytes, and fibroblasts. Oxygen radicals, such as superoxide (O2) and hydrogen peroxide, destroy nitric oxide and produce the toxic NO2 and peroxynitrite (1-3). Nitrogen dioxide causes pulmonary inflammation, lowers levels of lung antioxidants (9), including glutathione, destroys respiratory defense mechanisms, and increases susceptibility to respiratory pathogens and cancer (1,7). Nitrogen dioxide can also increase the incidence and severity of respiratory infections, can reduce lung function, and can aggravate the symptoms of asthmatics or subjects with COPD (1,8).
Peroxynitrite ion and peroxynitrous acid, formed from the interaction of nitric oxide and superoxide anions, hydroxyl radicals, and/or hydrogen peroxide, are strong oxidant species that work against nitric oxide by inducing single-strand breaks in DNA, increasing the levels of inflammatory mediators by activating NF-kappa-B and enhancing tumor formation and growth (21,64,65,71) rather than death. These properties have been demonstrated in Karposi's sarcoma in AIDS patients. Peroxynitrites are very toxic and disruptive to cell membranes via lipid peroxidation not only leading to cell death, but also dysfunction of many cellular membrane functions, such as transport mechanisms. Their effect can destroy the ability of white blood cells to kill invading microorganisms. Over-expression of peroxynitrite has been shown to destroy immune cells at the sites of infection, including CD4 and CD8 cells. Over-expression of peroxynitrite has been shown to enhance bacterial and viral replication at infected sites due to the ability of peroxynitrite to enhance NF-kappa-B expression. Peroxynitrites, which cause bronchial constriction, are involved in lung injury through the production of chemokines and contribute to viral pathogenesis and enhance viral mutations (2,3,13,30,65). Nitric oxide when combined with superoxide anions and/or hydrogen peroxide to form peroxynitrite, can also generate the highly reactive hydroxyl anion (OH), which lowers cellular levels of glutathione. The underlying chronic inflammatory process in wounds, which induces nitric oxide synthesis, also produces excess oxygen radicals, which will destroy nitric oxide (5,6). Infected and noninfected wounds enhance nitric oxide production by alveolar macrophages in rats, which also produces an increased level of oxygen radical that can react directly with nitric oxide to produce NO2 and peroxynitrites. (1-3,5,13) Peroxynitrites can also react with antimicrobial and anticancer drugs to destroy their ability to kill infections and cancer cells. Doxorubicin is a NF-kappa-B inducer, which dramatically increases the generation of peroxynitrite, which causes the damage produced by this drug to humans. The use of peroxynitrite inhibitors, like mercaptoethylguanidine, has been shown to reduce the damage from peroxynitrite allowing peroxynitrite treated cells to survive.
Nitric oxide, an oxidation product of nitrogen, is produced normally by many cell types, including endothelial cells and macrophages (1,2,3,12,15,16,17,26,27). Nitric oxide can act as a neurotransmitter, vasodilator, antibacterial, antiviral, and tumoricidal agent (12-18,72). Nitric oxide also possesses anti-inflammatory effects, which may be exerted via its ability to inhibit the transcription factor, NF-kappa-B (51) and other inflammatory cytokines (73). The most commonly proposed mode of action for the inhibition of NF-kappa-B involves interference with NF-kappa-B binding to DNA (53). Because IKK-β is subject to redox regulation, nitric oxide will inhibit NF-kappa-B activation by the inactivation of IKK β. High doses of nitric oxide also impaired the TNF-α-induced DNA binding activity of NF-kappa-B (55,64). High doses of nitric oxide also repressed the TNF-α induced transactivation by NF-kappa-BB (53-55) High doses of nitric oxide will inhibit NF-kappa-B. Nitric oxide also produces clinically useful bronchodilation (1) and is also used by the body to kill bacteria, fungal infections, viral infections, and tumors (21,72). Nitric oxide can kill these cell types because bacterial, viral, and tumor cells have no defenses against nitric oxide. Normal mammalian cells can cope with normal levels of nitric oxide by using enzyme systems to use or deactivate elevated cellular levels of nitric oxide (21-25). Nitric oxide is the main mediator of the tumoricidal action of activated macrophages (22-25,72).
While many papers have been written on nitric oxide, the role of nitric oxide in tumor biology was not completely understood until recently. Nitric oxide appeared to have both tumor promoting and inhibiting effects (24). Recent publications have implicated the reactive oxygen species made from nitric oxide during the inflammatory process, particularly peroxynitrite and nitrogen dioxide as being the tumor promoting agents, not nitric oxide itself (3,13,21,30). Nitric oxide does not mediate but inhibits transformation and tumor growth (72). Thus the ability to regulate the production of peroxynitrite would have tremendous therapeutic efficacy especially to protect drugs.
Sodium pyruvate, an α-keto acid, is an antioxidant that reacts directly with oxygen radicals like hydrogen peroxide and peroxynitrite, to neutralize them thereby protecting DNA and other cellular components, such as glutathione, lipids and proteins (35,56-65,70,71). In macrophages, and other cell lines, sodium pyruvate regulates the production and level of inflammatory mediators including oxygen radical production and also regulates the synthesis of nitric oxide (8,49,50). Sodium pyruvate has been administered to patients for a variety of medical disorders and applications including therapeutically and diagnostically in the treatment of Friedreich's ataxiai, and as a constituent in a therapeutic solution used in open heart surgery. It has been administered by several routes including intravenous, topical (for hyperkeratotic disorders), and oral (dietary supplements). In all cases, the administration of sodium pyruvate to these patients was shown to reduce inflammation and enhance healing. Pyruvate decreases the expression of several proinflammatory genes, including NF-kappa-B, activation of inducible nitric oxide synthase mRNA, TNF, cyclo-oxygenase, interleukin 6 and 10mRNA induction (32,33,44,49,50,64). Sodium pyruvate inhibited hydrogen peroxide induced transcription of NF-kappa-B while protecting cellular glutathione (44,64). Further, sodium pyruvate blocked the p38 MAPK pathway and activated the ERK pathway which regulates the expression of genes believed to prevent apoptosis and promote cell survival (44,64). Sodium pyruvate inhibited hepatocytes nitric oxide synthesis (27), and caused up regulation of inducible nitric oxide synthase mRNA in intestinal cells and in cardiac monocytes (8,26,28). It can specifically lower the overproduction of superoxide anions, H2O2 and nitric oxide in white blood cells (31,56-64).
Sodium pyruvate also increases cellular levels of glutathione, a major cellular antioxidant (8), needed to prevent activation of NF-kappa-B which activates the inflammatory process. It was recently discovered that glutathione is reduced dramatically in antigen-induced asthmatic patients (10) and inhaled glutathione does not readily enter cells. Pyruvate does enter all cells via a transport system and can also cross the blood brain barrier. Oxygen radicals are involved in the induction and progression of malignancy and pyruvate, a known scavenger of oxygen radicals, has been implicated in cancer prevention (32,33,57,64).
Pyruvate inhibited the growth of implanted tumors and reduced lung metastases and decreased the number of DNA breaks caused by H2O2 by 40% (32). Excess sodium pyruvate, beyond that needed to neutralize oxygen radicals, will enter the mammalian cells. All cells have a transport system that allow cells to concentrate pyruvate at higher concentrations than serum levels. In monocytes cultures, the production of H2O2 was regulated by the level of sodium pyruvate supplied in the culture medium. At 1 mM and higher concentrations, the levels of H2O2 was decreased by 30%. At 10 mM concentrations, the levels of H2O2 was decreased by 60% (64).
Pyruvate controls the positive and negative effects of nitric oxide at higher levels. Too high a level of nitric oxide is detrimental to cells. When higher levels of nitric oxide are produced, even by activation of inducible nitric oxide synthase mRNA from higher levels of pyruvate, it is kept in control by pyruvate. Nitric oxide affects cells by increasing levels of cGMP and ADP (adenosine diphosphate), and requires an acid pH range in which to work (12). Higher levels of pyruvate raises the pH level, increases levels of ATP, decreasing levels of ADP and cAMP, and increases levels of GTP, while decreasing levels of cGMP. Thus pyruvate will protect cells from excess nitric oxide. Increased nitric oxide levels are chemotactic for eosinophils, which produce and enhance inflammation (13), especially if it is transformed into peroxynitrite. Eosinophils affect dyspnoea perception in asthma by releasing neurotoxins (13).
Inflammation is a nonspecific response caused by a variety of injuries including the penetration of the host by an infectious agent. The distinguishing feature of inflammation is the dilation and increased permeability of minute blood vessels. The inflammatory response consists of three successive phases: (a) increased vascular permeability with resulting edema, pain, and swelling, (b) cellular infiltration and phagocytoses, and (c) proliferation of the fibroblasts synthesizing new connective tissue to repair the injury. A large number of mediators of inflammation have been implicated in the inflammatory process primarily in terms of their capacity to induce vasodilation and increased permeability. Inflammation also increases levels of compounds that increase pain, erythema, ischemia, excess angiogenesis, swelling, crusting, itching, and scarring.
Direct injury, such as that caused by toxins produced by microorganisms, leads to destruction of vascular endothelium and results in the increased permeability to plasma proteins, especially in the venules and venular capillaries. Mediators of secondary injury are liberated from the site of direct injury. As a result, gaps form between vascular endothelial cells through which plasma proteins escape. Granulocytes, monocytes, and erythrocytes may also leave vascular channels. Mediators of secondary injury include unknown substances and histamine, peptides (kinins), kinin-forming enzymes (kininogenases), and a globulin permeability factor. These mediators are blocked from action by antihistamines and sympathoamines, and are most pronounced in effect on venules, although lymph-vascular endothelium also becomes more porous as a part of secondary injury. In the early stages of inflammation, the exudate is alkaline and neutrophilic polymorphonuclear leukocytes predominate. As lactic acid accumulates, presumably from glycolysis, the pH drops and macrophages become the predominant cell type. Lactic acid and antibodies in the inflammatory exudate may inhibit parasites, but the major anti-infectious effect of the inflammatory response is attributable to phagocytic cells.
The beneficial effect of the inflammatory response is the production of: (1) leukocytes in great numbers; (2) plasma proteins, nonspecific and specific humoral agents, fibrinogen that on conversion to fibrin aids in the localization of the infectious process while acting as a matrix for phagocytoses; and (3) increased blood and lymph flow that dilutes and flushes toxic materials while causing a local increase in temperature.
The initial increase in capillary permeability and vasodilation in an inflamed wound is followed by an increase in metabolism of the tissues. Leakage of fibrinogen into the wound, where proteolytic enzymes convert it into fibrin thrombi, establishes a capillary and lymphatic blockade. The concentrations of components of the ground substance of connective tissue collagen, mucopolysaccharides, glycoproteins, and nonfibrous proteins are greatly increased during this process. As the exudative phase of the inflammation subsides, the fibroblast is found to be the dominant cell in the wounded zone. The fibroblast first proliferates, then synthesizes extracellular material, including new collagen fibers and acid mucopolysaccharides, which are laid down to form the new tissue matrix.
On a macroscopic level, the inflammatory phenomenon is usually accompanied by the familiar clinical signs of erythema, swelling, edema tenderness (hyperalgesia), and pain. During this complex response, chemical mediators such as histamine, 5-hydroxytryptamine (5-HT), slow-reacting substance of anaphylaxis (SRS-A), various chemotactic factors, bradykinin, and prostaglandins are liberated locally. Phagocytic cells migrate into the area, and cellular lysosomal membranes may be ruptured, releasing lytic enzymes. All these events may contribute to the inflammatory response.
The production of reactive oxygen intermediates has been suggested to cause many skin, tissue, and organ disorders such as atherosclerosis, arthritis, cytotoxicity, skin inflammation, photoaging, wrinkling, actinic keratosis, tumor formation, cancer, hypertension, Parkinson's Disease, lung disease, and heart disease. The role of active oxygen radicals in promoting tumors has been based on the findings that (a) tumor promoters increase the level of oxygen radicals, (b) many free radical-generating systems promote tumors, and (c) certain antioxidants inhibit the biochemical effects of tumor promoters.
In vitro, reactive oxygen intermediates can be generated in cellular culture media by auto-oxidation and photo-oxidation of media components. During excision and storage, transplant organs can suffer oxidative injuries which result in the loss of cellular membrane integrity and shorten the usable life of the organ.
When cells are stressed by oxidative injury, a resuscitation step is necessary to re-condition the cells. Anti-oxidants have been shown to inhibit damage associated with active oxygen species. For example, pyruvate and other α-keto acids have been reported to react rapidly and stoichiometrically with hydrogen peroxide to protect cells from adverse cytolytic effects (61).
U.S. Pat. No. 5,210,098, issued to Nath, disclose a method to arrest or prevent acute kidney failure by administration of a non-toxic pyruvate salt to a patient in need of such treatment. Nath discloses a therapeutic method comprising the administration of an amount of a pyruvate salt to a patient experiencing or in danger of, acute renal failure. The pyruvate salt, preferably sodium pyruvate, is dispersed or dissolved in a pharmaceutically acceptable liquid carrier and administered parenterally in an amount effective to arrest or prevent the acute renal failure, thus permitting restoration of normal kidney function. In some cases, the pyruvate may be infused directly into the kidney or into the proximal renal arterial circulation. The method is effective to prevent or counteract acute kidney failure due to a wide variety of causes, including, but not limited to, traumatic injury including burn injury and obstruction; reperfusion following ischemia, inflammatory glomerulonephritis, and sepis, e.g., due to gram negative bacterial infection.
U.S. Pat. No. 5,296,370, issued to Martin, et al., discloses therapeutic compositions for preventing and reducing injury to mammalian cells and increasing the resuscitation rate of injured mammalian cells. In one embodiment, the therapeutic composition comprises (a) a pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the resuscitation of injured mammalian cells.
U.S. Pat. No. 5,256,697, issued to Miller, et al., discloses a method for orally administering a therapeutically effective amount of a pyruvate precursor to a mammal to improve insulin resistance, lower lasting insulin levels and reduce fat gain.
U.S. Pat. Nos. 3,920,835; 3,984,556, and 3,988,470, all issued to Van Scott, et al. disclose methods for treating acne, dandruff, and palmar keratosis, respectively, which consist of applying to the affected area a topical composition comprising from about 1% to about 20% of a lower aliphatic compound containing from two to six carbon atoms selected from the group consisting of α-hydroxy acids, α-keto acids and esters thereof, and 3-hydroxybutryic acid in a pharmaceutically acceptable carrier. The aliphatic compounds include pyruvic acid and lactic acid.
U.S. Pat. Nos. 4,105,783 and 4,197,316, both issued to Yu, et al., disclose a method and composition, respectively, for treating dry skin which consists of applying to the affected area a topical composition comprising from about 1% to about 20% of a compound selected from the group consisting of amides and ammonium salts of α-hydroxy acids, β-hydroxy acids, and α-keto acids in a pharmaceutically acceptable carrier. The compounds include the amides and ammonium salts of pyruvic acid and lactic acid.
U.S. Pat. No. 4,234,599, issued to Van Scott, et al., discloses a method for treating actinic and non-actinic skin keratoses which consists of applying to the affected area a topical composition comprising an effective amount of a compound selected from the group consisting of α-hydroxy acids, β-hydroxy acids, and α-keto acids in a pharmaceutically acceptable carrier. The acidic compounds include pyruvic acid and lactic acid.
U.S. Pat. No. 4,294,852, issued to Wildnauer, et al., discloses a composition for treating skin which comprises the α-hydroxy acids, β-hydroxy acids, and α-keto acids disclosed above in combination with C3-C8 aliphatic alcohols.
U.S. Pat. No. 4,663,166, issued to Veech, discloses an electrolyte solution which comprises a mixture of L-lactate and pyruvate in a ratio from 20:1 to 1:1, respectively, or a mixture of D-β-hydroxybutyrate and acetoacetate, in a ratio from 6:1 to 0.5:1, respectively.
Sodium pyruvate has been reported to reduce the number of erosions, ulcers, and hemorrhages on the gastric mucosa in guinea pigs and rats caused by acetylsalicylic acid. The analgesic and antipyretic properties of acetylsalicylic acid were not impaired by sodium pyruvate, Puschmann, Arzneimittelforschung, 33, pp. 410-416 (1983).
Pyruvate has been reported to exert a positive inotropic effect in stunned myocardium which is a prolonged ventricular dysfunction following brief periods of coronary artery occlusions which does not produce irreversible damage, Mentzer, et al., Ann. Surg., 209, pp. 629-633 (1989). Pyruvate has also been reported to produce a relative stabilization of left ventricular pressure and heart work parameter and to reduce the size of infarctions. Pyruvate improves resumption of spontaneous beating of the heart and restoration of normal rates and blood pressure development, Bunger, et al., J. Mol. Cell. Cardiol., 18, pp. 423-438 (1986), Mochizuki, et al., J. Physiol. (Paris), 76, pp. 805-812 (1980), Regitz, et al., Cardiovasc. Res., 15 pp. 652-658 (1981), Giannelli, et al., Ann. Thorac. Surg., 21 pp. 386-396 (1976).
Sodium pyruvate has been reported to act as an antagonist to cyanide intoxication (presumably through the formation of cyanohydrin) and to protect against the lethal effects of sodium sulfide and to retard the onset and development of functional, morphological, and biochemical measures of acrylamide neuropathy of axons, Schwartz, et al., Toxicol. Appl. Pharmacol., 50 pp. 437-442 (1979), Sabri, et al., Brain Res., 483, pp. 1-11 (1989).
U.S. Pat. No. 5,798,388, issued to Katz, discloses a method and compositions for the treatment of pulmonary diseases resulting from inflammation consisting of the administration of pyruvate, lactate, and precursor thereof and their salts in a pharmaceutically acceptable carrier. The compositions may also be a cellular energy source.
A chemotherapeutic cure of advanced L1210 leukemia has been reported using sodium pyruvate to restore abnormally deformed red blood cells to normal. The deformed red blood cells prevented adequate drug delivery to tumor cells, Cohen, Cancer Chemother. Pharmacol., 5, pp. 175-179 (1981).
Primary cultures of heterotopic tracheal transplant exposed in vivo to 7, 12-dimethylbenz(a)anthracene were reported to be successfully maintained in enrichment medium supplemented with sodium pyruvate along with the cultures of interleukin-2 stimulated peripheral blood lymphocytes, and plasmacytomas and hybridomas, pig embryos, and human blastocysts, Shacter, J. Immunol, Methods, 99, pp. 259-270 (1987), Marchok, et al., Cancer Res., 37, pp. 1811-1821 (1977), Davis, J. Reprod. Fertil, Suppl., 33, pp. 115-124 (1985), Okamoto, et al., No To Shinkei, 38, pp. 593-598 (1986), Cohen, et al., J. In vitro Fert. Embryo Transfer, 2, pp. 59-64 (1985).
U.S. Pat. Nos. 4,158,057; 4,351,835; 4,415,576, and 4,645,764, all issued to Stanko, disclose methods for preventing the accumulation of fat in the liver of a mammal due to the ingestion of alcohol, for controlling weight in a mammal, for inhibiting body fat while increasing protein concentration in a mammal, and for controlling the deposition of body fat in a living being, respectively. The methods comprise administering to the mammal a therapeutic mixture of pyruvate and dihydroxyacetone, and optionally riboflavin. U.S. Pat. No. 4,548,937, issued to Stanko, discloses a method for controlling the weight gain of a mammal which comprises administering to the mammal a therapeutically effective amount of pyruvate, and optionally riboflavin. U.S. Pat. No. 4,812,479, issued to Stanko, discloses a method for controlling the weight gain of a mammal which comprises administering to the mammal a therapeutically effective amount of dihydroxyacetone, and optionally riboflavin and pyruvate.
Rats fed a calcium-oxalate lithogenic diet including sodium pyruvate were reported to develop fewer urinary calculi (stones) than control rats not given sodium pyruvate, Ogawa, et al., Hinvokika Kivo, 32, pp. 1341-1347 (1986).
U.S. Pat. No. 4,521,375, issued to Houlsby, discloses a method for sterilizing surfaces which come into contact with living tissue. The method comprises sterilizing the surface with aqueous hydrogen peroxide and then neutralizing the surface with pyruvic acid.
U.S. Pat. No. 4,416,982, issued to Tauda, et al., discloses a method for decomposing hydrogen peroxide by reacting the hydrogen peroxide with a phenol or aniline derivative in the presence of peroxidase.
U.S. Pat. No. 4,696,917, issued to Lindstrom, et al., discloses an irrigation solution which comprises Eagle's Minimum Essential Medium with Earle's salts, chondroitin sulfate, a buffer solution, 2-mercaptoethanol, and a pyruvate. The irrigation solution may optionally contain ascorbic acid and α-tocopherol.
U.S. Pat. No. 4,725,586, also issued to Lindstrom, et al., discloses an irrigation solution which comprises a balanced salt solution, chondroitin sulfate, a buffer solution, 2-mercaptoethanol, sodium bicarbonate or dextrose, a pyruvate, a sodium phosphate buffer system, and cystine. The irrigation solution may optionally contain ascorbic acid and gamma-tocopherol.
U.S. Pat. No. 4,847,069, issued to Bissett, et al., discloses a photoprotective composition comprising (a) a sorbohydroxamic acid, (b) an anti-inflammatory agent selected from steroidal anti-inflammatory agents and a natural anti-inflammatory agent, and (c) a topical carrier. Fatty acids may be present as an emollient.
U.S. Pat. No. 4,847,071, also issued to Bissett, et al., discloses a photoprotective composition comprising (a) a tocopherol or tocopherol-ester radical scavenger, (b) an anti-inflammatory agent, and (c) a topical carrier.
U.S. Pat. No. 4,847,072, issued to Bissett, et al., discloses a topical composition comprising not more than 25% tocopherol sorbate in a topical carrier.
U.S. Pat. No. 5,863,938, issued to Martin, discloses a therapeutic antibacterial wound-healing composition comprising an effective amount of an antibacterial agent and a wound-healing composition consisting of (a) pyruvate- or α-keto-glutaric acid (b) an antioxidant, and (c) a mixture of fatty acids.
U.S. Pat. No. 5,561,157, issued to Yu, et al., discloses a composition and method for the therapeutic treatment of age spots, wrinkles, dry skin, eczema, psoriasis, and keratosis, using α- and β-keto-carboxylic acids and their salts.
U.S. Pat. No. 6,149,924, issued to Paul, discloses the use of many agents that increase the production of skin lipids, increase barrier function, hydrogen peroxide neutralization, prevention of loss of moisturizing factor from the skin. The agents are amino acids and their breakdown products.
U.S. Pat. No. 5,633,285, issued to Martin, discloses a therapeutic cytoprotective wound healing composition. The composition comprises a cytotoxic agent and a therapeutic wound healing composition which comprises (a) pyruvate (b) vitamin E, and (c) a mixture of saturated and unsaturated fatty acids. The invention is used to protect normal cells from cytotoxic drugs used in the treatment of cancer.
U.S. Pat. No. 5,536,751, issued to Bunger, discloses a pharmaceutical composition as an active phosphorylation potential enhancing substance using an α-keto-carboxylic acid, primarily pyruvate.
U.S. Pat. No. 6,689,810, issued to Martin, discloses a method for treating pulmonary disease state in mammals by altering indigenous in vivo levels of nitric oxide in mammalian cells, using α-keto acids.
U.S. patent application No. 20030165457 (Martin) discloses a method for treating wounds, injuries, diseases and dermatological disease states in mammals caused by mammalian cells involved in the inflammatory response comprising contacting the mammalian cells with an antioxidant reactive oxygen species mediator selected from the group consisting of α-keto acids used singly or in combination in an amount capable of reducing the undesired inflammatory conditions.
The addition of sodium pyruvate to bacterial and yeast systems has been reported to inhibit hydrogen peroxide production, enhance growth, and protect the systems against the toxicity of reactive oxygen intermediates. The optimum ratio of unsaturated to saturated fatty acids contained within chicken fat enhanced membrane repair and reduced cytotoxicity. The anti-oxidants gluthathione and thio-glycollate reduced the injury induced by oxygen radical species.
While the above therapeutic compositions and methods are reported to act as antioxidants that neutralize the negative effects of reactive oxygen radicals, none of the compositions and methods treat the damage and resulting disease state in mammals caused by proliferative, degenerative, cancer and infected and non-infected wounds by altering indigenous in vivo levels of peroxynitrite in mammalian cells, while protecting cells from the toxic metabolites produced by peroxynitrite. None of the therapeutic methods have devised a way to protect the drugs needed to treat these various diseases and increase their efficacy and duration. Drugs such as antivirals, antibacterials, antifungals, antitelomerases and anticancer drugs when attacked by oxygen radicals especially peroxynitrite and various inflammatory mediators controlled by NF-kappa-B will become cytotoxic compounds that will further enhance the activation of NF-kappa-B. The healing process requires the inhibition and control from the production of peroxynitrite and the reversal of cytotoxicity from the catabolism of drugs and the elimination of metabolic toxic by-products, the suppression of inflammation, including the over-expression of NF-kappa-B by these toxic by-products, the stimulation of cellular viability and proliferation. Healing also requires compounds that react directly or indirectly with toxic agents to inhibit their activation of NF-kappa-B. Patients who suffer major wounds could benefit from decreasing the over-expression of peroxynitrite and other inflammatory mediators controlled by NF-kappa-B to protect and enhance repair with and without the use medicines to reduce the pain, swelling, tissue ischemia, excess angiogenesis, erythema (redness), crusting, itching, and fibrotic conditions (scarring) which accompany most infected and non-infected wounds and cancer. This therapy will reduce undesired pain, progressive tissue ischemia, excess angiogenesis, excess white blood cell (WBC) infiltration, erythema, swelling, itching, crusting, and scarring. Moreover, cellular signaling agents in mammalian cells are needed to deposit the correct ratio and type of collagen and elastin.